Methods for cancer imaging

ABSTRACT

Methods are provided for cancer and pre-cancer detection by increased uptake of fluorophore glucose or deoxyglucose conjugates in cancerous and pre-cancerous cells relative to normal cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to provisional U.S. patent applicationSer. No. 60/342,313, filed 21 Dec. 2001, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The term “cancer” generally refers to one of a group of more than 100diseases that are caused by the uncontrolled growth and spread ofabnormal cells and can take the form of solid tumors, lymphomas andnon-solid cancers such as leukemia Unlike normal cells, which reproduceuntil maturation is attained and then only-reproduce as necessary toreplace wounded cells, cancer cells grow and divide endlessly withoutdifferentiating to mature, functional cells, crowding out nearby cellsand eventually spreading to other parts of the body.

The most common sites in which cancer develops include the skin, lungs,female breasts, prostate, colon, rectum, bladder, uterus, blood-formingtissues, and lymphatic system. Cancer cells that have developed at oneof these sites will grow rapidly into a malignant tumor, invading anddestroying nearby tissues. Malignant cancer tumors will eventuallymetastasize, or spread to other parts of the body, unless theirprogression is stopped.

Cancers are easier to treat and cure if they are discovered and treatedprior to metastasis. The survival of a patient with cancer is generallyinfuenced by the stage at which the cancer is diagnosed. The stage,generally categorized as 1-4, is determined by the extent of disease,with stage 1 cancers being those that are small and not invading thesurrounding tissues, while stage 4 cancers have established tumors intissues other than the organ in which the cancer arose. Once cancercells metastasize by leaving a tumor, they will travel through thebloodstream or lymphatic system to other parts of the body, where thecells begin multiplying and developing into new tumors. This sort oftumor progression makes cancer dangerously fatal. Although there havebeen great improvements in diagnosis, general patient care, surgicaltechniques, and local and systemic adjuvant therapies, most deaths fromcancer are still due to metastases that are either resistant toconventional therapies or are undetected by current diagnostic methods.

The majority of diagnostic methods depend on microscopic observation oftissue biopsies. Many biopsy methods are invasive and require surgicalremoval of tissue for analysis.

Other useful methods for detecting cancer tissue in an animal includethe use of positron emission tomography (PET scan). This method takesadvantage of the increased uptake and retention by malignant cells ofglucose and uses an ¹⁸F-labeled glucose derivative (FDG,¹⁸F-2-fluoro-2-deoxyglucose). This glucose derivative, lacking a hydroxygroup at the 2-position, cannot be further metabolized by the cells andis simply accumulated in the cells. This method of detection revealslive, growing tumor cells and therefore has advantages over anatomicdetection methods which show abnormal structures but are insensitive tothe viability of these abnormal tissues. While PET scanning is quiteuseful to image the entire patient for cancer, it requires expensive andcumbersome equipment for detection and construction of the positron usedfor the image. Accordingly, PET scanning cannot be used for thedetection of cancer in many clinical settings, such as in thephysician's office during the time of physical examination or in theoperating room at the time of surgery.

Detection of a skin cancer such as melanoma has typically been throughphysical examination of the skin followed by biopsy of selected lesionssuspected to be cancerous. Drawbacks to this procedure reside in theexperience of the examiner, and errors in diagnosis can be lifethreatening. In instances where cancers are missed and then spreadbeyond the original site of disease, mortality can increase. Conversely,some skin lesions are biopsied which are not cancerous, and the patientsare thus subjected to unnecessary harm. In some cases, biopsy samplesare taken from the face, leading to cosmetic debility.

For those instances in which a patient has been diagnosed with cancer,the physician generally determines if the cancer can be removed, orresected, by surgery. Patients who have cancer that has not spreadbeyond a local area, stage 1 or 2, frequently may be cured by completelyresecting the tumor. Prior to the surgery, various images of the tumorare obtained such as X-rays, CT scans, MRI scans or PET images. Thesetests provide guidance for surgery, but at the time of surgery, theseimages cannot be generated in real time to guide the surgeon to thetumor. As a result, the surgeon must use the unaided senses of sight andfeel to determine the location and extent of the tumor.

In some instances, the surgeon will obtain biopsy samples in the area ofthe resected tumor prior to completing the operation. These samples areexamined under the microscope to determine if all of the cancer has beenremoved. However, this procedure is often not conducted, as it requiresa highly trained pathologist to be present at the surgery and to rapidlyanalyze the tissue sample while the patient remains on the operatingtable. If this analysis is used and the cancer remains in the patient,the surgeon continues with unaided senses to try to resect any residualtumor. Unfortunately, despite such efforts, residual tumor will be leftinside the patient about 15-25% of the time. Studies have shown thatthese patients are at greater risk of dying of the cancer than thosethat have the tumor completely resected. Because of this, these patientsrequire further, often debilitating, costly therapy in an attempt toarrest and treat the cancer left in the patient at the time of surgery.

Screening for tumors in the colon or lung is currently carried out byendoscopy using white light and a video capture screen. Due to thenative fluorescence of lung tumor tissue, a special adapter for theendoscope is used to detect this autofluorescence, thereby enabling theobserver to detect smaller tumors at an earlier time. However, even withthis enhanced method of screening, smaller tumors and cancer cells cango undetected.

To image cancer tissue at the time of screening or at the time ofsurgery, attempts have been made to use radioactive isotopes orphotosensitizers linked to targeting entities such as monoclonalantibodies. These detection methods are limited in that they cannot beused in the general physician's practice for screening large numbers ofpatients nor can they be used at the time of surgery to locate theresidual tumor. Other detection methods are described in U.S. Pat. Nos.6,256,530, 6,091,985, 6,083,487; EP patent publication No. 0588994 A1;and PCT patent publication Nos. WO 96/10363 and WO 93/13403.

What is needed are methods and reagents for detecting cancer tissue,including tumors, non-solid cancers, and cancer cells, that areconvenient, without the drawbacks of the methods and reagents notedabove, and that are widely applicable in a variety of clinical settings,including surgery. The present invention provides such methods, as wellas compounds and compositions useful in the methods.

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions and methods fordetecting and imaging cancerous and pre-cancerous cells and tissues.

In one aspect, the present invention provides methods for detecting orimaging cancer in a subject, comprising:

(a) administering to the subject an effective amount of a fluorophoreglucose or deoxyglucose conjugate; and

(b) detecting or imaging cells that take up the fluorophore conjugate orderivative to determine cancerous or pre-cancerous cells or tissue arepresent in the subject.

In one embodiment, the fluorophore conjugate has the formula:Fl-L-Glcwherein Fl is a fluorophore; L is a bond or a linking group; and Glc isglucose, deoxyglucose, or a glucose or deoxyglucose derivative.Preferred glucose derivatives are D-(+)-deoxyglucose, D-(+)-glucosamineand N-acetyl D-glucosamine. The above method can be carried out incombination with a surgical procedure, such as a cancer resection. Themethod of detecting can be carried out endoscopically for example, orvisually, for example as part of a skin examination for melanomascreening.

Additionally, this method finds broad applicability to the detection ofa number of cancers, due in large part to its ability to detect and/orimage cancer at the cellular level from such cancers as lung cancer,breast cancer, prostate cancer, colon cancer, cervical cancer,esophageal cancer, bladder cancer, head and neck cancer, and melanoma.

In some embodiments, additional steps can be employed, such as apreliminary step of reducing glucose ingestion in a subject prior toadministering the fluorophore glucose or deoxyglucose conjugate.Typically, this can be accomplished by withholding carbohydrateconsumption for a period of up to 18 hours prior to administration ofthe conjugate. In other embodiments, carbohydrate consumption is stoppedfor a period of 8 to 48 hours prior to administration of the conjugate.

In a related aspect, the present invention provides methods fordetecting or imaging precancerous cells in a subject, comprising:

(a) administering to the subject an effective amount of a fluorophoreglucose or deoxyglucose conjugate; and

(b) detecting or imaging pre-cancerous cells that take up thefluorophore glucose or deoxyglucose conjugate to determine ifpre-cancerous cells are present in the subject.

In another related aspect, the present invention provides a method forcancer or pre-cancer detection during an operative or endoscopicprocedure, the method comprising:

(a) administering to a patient subject to such a procedure, an effectiveamount of a fluorophore glucose or deoxyglucose conjugate, the conjugatehaving a rate of uptake in cancerous or pre-cancerous cells that is atleast two times greater than the rate of uptake in normal cells;

(b) conducting the procedure within 48 hours of administering theconjugate; and

(c) scanning the patient with a detection means for detecting thelocalization of the fluorophore conjugate in the cancerous orpre-cancerous cells.

In preferred embodiments of this aspect, the procedure is conductedwithin about 24 hours of administering the conjugate. More preferably,the method further comprises treating sites of conjugate accretion byexternal beam radiation, laser therapy or surgical removal.

In yet another aspect, the present invention provides a fluorophoreglucose or deoxyglucose conjugate having the formula:Fl-L-Glcwherein Fl is a fluorophore having an emission wavelength of from about400 nm to about 1200 nm; L is a bond or a linking group; and Glc isglucose or deoxyglucose or a glucose or deoxyglucose derivative, withthe proviso that the conjugate is other than2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG).

The present invention further provides kits comprising a container witha sterile preparation for human use of a fluorophore glucose ordeoxyglucose conjugate and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the results of a time-course uptake of2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in Rajilymphoma cells.

FIG. 2 is a histogram providing a comparison of2-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake inRaji lymphoma cells versus non-cancer PBMC cells (FIG. 2A) and where thesignals are normalized for the presence of red blood cells in PBMC (FIG.2B).

FIG. 3 is a graph illustrating the results of a time-course uptake ofFGC-002 relative to2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in Rajilymphoma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention arises in part from the discovery that cancerousand pre-cancerous cells exhibit an enhanced rate of uptake of glucosefluorophore (fluorescent) conjugates.

The present invention makes possible the imaging of tumor tissue duringscreening visits to a doctor and at the time of surgery by usingfluorophore glucose or deoxyglucose conjugates. Some of these conjugatesare derived by chemically modifying 2-deoxyglucose or an analog thereof,which is taken into and accumulated in cancerous cells and pre-cancerouscells (e.g., dysplastic cells) preferentially, compared to normal cells.After allowing the fluorophore glucose conjugate to accumulate in thecancerous cells, an examination can be made of the cancer or of thepatient with either a camera or other device to view or capture thefluorescence. This method allows cancerous tissue to be detected morespecifically and with greater sensitivity than existing methods ofdetection.

In one embodiment of the present invention, the method is used by asurgeon to visualize, in real time, tumor tissue during a surgicalresection and to assess the margin of the tumor, allowing a morecomplete resection of the tumor tissue and a better outcome for thepatient.

Additionally, administration of the fluorophore conjugate to a humanprior to examination can be carried out to determine if the humansubject has cancerous or pre-cancerous tissue in any observed organ. Forexample, an individual who is at high risk for developing melanomabecause of skin type and family history can be administered afluorophore conjugate prior to examination. After accumulation of theconjugate in any tumor tissue, visualization of the tumor can be madewith appropriate equipment. In addition to standard methods ofexamination, a diagnosis can be made by determining if any of thepigmented lesions on the skin are cancerous and must be removed. Thepresent invention eliminates many errors of diagnosis that can lead toeither the unneeded removal of benign tissue or the mistake of leaving acancer on the skin, allowing it to spread throughout the body.

The methods of the present invention are useful in the course ofsurgery, visual examinations, endoscopic examinations, and the like.Typically, the methods will involve contacting sites on a subjectsuspected of being cancerous sites with a suitable fluorophoreconjugate, and visualizing the cells and tissues that have taken up theconjugate to determine in real time whether cancerous or pre-cancerouscells are present in the subject.

The methods described herein do not require the processing of images butinstead can be carried out by the clinician or surgeon with the use of,e.g., an intraoperative probe, an intravascular probe, or an endoscopeto scan areas of suspected cancerous cells or tissue and to correlatethe level of fluorescence observed with the presence or absence ofcancerous cells and to discriminate cancerous cells and tissue fromnon-cancerous cells and tissue more precisely. Using this method, thesurgeon or clinician can more precisely define tumor borders forsurgical resection or diagnostic evaluation. Additionally, drugdelivery, laser therapy, radiation therapy, external beam therapy, andthe like can be more specifically targeted to cancerous cells andtissues harboring them that have been identified by the methodsdescribed herein.

Methods of Detecting or Imaging Cancer

In view of the above, the present invention provides, in one aspect,methods for detecting or imaging cancerous cells or tissue in a subject,comprising:

(a) administering to the subject an effective amount of a fluorophoreglucose or deoxyglucose conjugate; and

(b) detecting or imaging cells that take up the fluorophore conjugate todetermine if cancer is present in the subject.

The present methods find broad applicability in the detection of anumber of cancers, due in large part to their utility in detectingcancer at the cellular level from all types of cancers, including butnot limited to such cancers as lung cancer, breast cancer, prostatecancer, colon cancer, cervical cancer, esophageal cancer, bladdercancer, head and neck cancer, melanoma, low grade non-Hodgkin'sLymphoma, intermediate grade non-Hodgkin's Lymphoma, follicularlymphoma, large cell lymphoma, B-cell lymphoma, T-cell lymphoma, Mantlecell lymphoma, Burkitt's lymphoma, NK cell lymphoma, and acutelymphoblastic lymphoma.

The fluorophore conjugate used in this aspect of the invention ispreferably one having the formula:Fl-L-Glc   (1)wherein Fl is a fluorophore; L is a bond or a linking group; and Glc isglucose or deoxyglucose or a glucose or deoxyglucose derivative.Fluorophores

A variety of fluorophores are useful in this aspect of the invention,including certain commercially available fluorophores such asfluorescein derivatives (e.g., fluorescein isothiocyanate), rhodaminederivatives and derivatives of the chelates of rare earth metals such aseuropium.

Selection of a suitable fluorophore generally involves consideration ofcertain physical and chemical properties such as fluorescence intensity,fluorescence lifetime, excitation and emission wavelength maxima,polarization, and non-specific binding behavior.

(a) Fluorescence Intensity

Fluorescence intensity is the intensity of the fluorescence producedupon excitation of the fluorophore with light (such as from a lasersource) and can be dependent on the nature of the solvent used for thefluorescence measurement. Use of an aqueous solvent system, such as abiological buffer, is convenient and preferred for certain applications,such as immunoassays.

(b) Excitation and Emission Wavelengths

The excitation and emission wavelengths are, respectively, thewavelengths of light required to produce fluorescence and thewavelengths of light at which fluorescence emission occurs. Fluorescenceemission occurs at a longer wavelength than the excitation wavelength.Ultraviolet, visible, and infrared light (typically wavelengths in therange of about 200 nanometers to about 1000 nanometers) are consideredto be wavelengths that are potentially useful in exciting a fluorophoremolecule and thereby producing detectable fluorescence. Due to theabundance of naturally occurring substances which fluoresce uponexcitation at relatively short wavelengths (in the range of about 200 nmto about 500 nm), improved sensitivity of detection can be achieved byusing a conjugate having a fluorophore which, in the conjugate,fluoresces upon excitation by light of wavelength greater than about 500nm, preferably in the spectral range of about 500 nm to about 900 nm.

Those of skill in the art will be able to select a conjugate of theinvention having the desired properties for the particular applicationof interest to the practitioner. There are a variety of fluorophoresthat can be selected to avoid or minimize the background signal fromautofluorescence of the surrounding tissue or fluids or to enhancefluorescence from a fluorophore present in cancerous as opposed tosurrounding benign tissue. Thus, as but one example, U.S. Pat. No.5,131,398, to Alfano et al., describes diagnosis of cancerous cells byusing a substantially monochromatic excitation light and two detectionbands at ˜340 and 440 nm. The patent reports that, when tissue ismonochromatically excited at 300 nm, the resulting native fluorescencespectrum from 320 nm to 600 nm in cancerous tissue is substantiallydifferent from that of either benign or healthy tissue. The patentfurther disclosed that, by avoiding the use of fluorescent emissionsbetween about 380 nm and 430 nm, the fluorescent effect from blood couldbe ignored. In addition, the patent reported that, at excitationwavelengths above 315 nm, the ratios of fluorescent-emission intensitiesare indistinguishable between cancerous and benign cells. The diversefluorophore deoxyglucose conjugates of this invention allow thepractitioner to select the conjugate best suited to detect cancer cellsin the tissue or tissues of interest given such parameters.

(c) Fluorescence Lifetime and Fluorescence Decay Time

The lifetime of the fluorescence produced by the fluorophore may varyfrom less than one nanosecond to several milliseconds. Some organic dyesthat exhibit fluorescence lifetimes in the range of 3 to 50 nanosecondsbelong to the general class of aromatic compounds, exemplified byaromatic hydrocarbon derivatives such as perlene carboxylic acid andaromatic heterocyclic compounds such as phthalocyanines and naturallyoccurring porphyrins. These dyes have a characteristic fluorescencelifetime, that is, the time period following excitation during whichthey emit light and during which the fluorescence intensity decreases toabout 37% (1/e) of its initial value in the absence of any deactivatingfactors. The measured fluorescence decay time is the time period duringwhich the decrease to the 37% (1/e) level of fluorescence intensity isobserved. The measured decay time of a particular compound may besolvent dependent. Under conditions which minimize deactivation,measured decay time may approach fluorescence lifetime.

(d) Fluorescence Polarization

When a fluorescent substance in solution is excited with polarizedlight, it emits partially polarized light as fluorescence. The degree ofpolarization of fluorescence can be measured and is related to themolecular volume of the fluorophore. Accordingly, generally preferredfluorophores are those which efficiently fluoresce upon excitation withlight whose wavelength falls within the range of about 200 to about 1000nanometers, preferably in the range of about 600 to 800 nanometers.Suitable fluorophores include those which absorb and/or emit atwavelengths which are distingtushable from the excitation and emissionmaxima of the other solution components (such as proteins present in asample) to minimize background fluorescence. For those methods involvingdetection or imaging during surgery, fluorophores having excitationand/or emission wavelengths of at least about 500 nanometers arepreferred. The use of such fluorophores reduces interference from theambient fluorescence of other biological components. Preferredfluorophores also exhibit a high degree of fluorescence polarization,preferably greater than about 10% of the theoretical maximum value foran observable polarization.

Preferred fluorophore moieties include fluorescent dyes having (a) highfluorescence intensity, (b) sufficiently long excitation and emissionwavelength maxima so that interference from natural fluorescence ofeither diseased or normal tissue is minimized; (c) sufficiently longmeasured fluorescence decay time to allow accurate measurement ofemitted light over background fluorescence and scattering (at leastabout 2, preferably at least about 10 nanoseconds); and (d) high degreeof fluorescence polarization. Additionally, in one embodiment of theinvention, a fluorophore glucose, deoxyglucose or derivative conjugatethat is transportable by the glucose transporter is provided and ispreferred in some applications of the methods of the invention. In thisembodiment, the preferred fluorophore has the additional quality ofsmall size.

Preferred fluorophores include, for example, 5-(and 6)carboxynaphtofluorescein and Texas Red, as described in the examplesbelow, especially for conjugation to the terminal primary amino group ofa linker attached to glucosamine. Additionally, other preferredfluorophores are: (i) BODIPY 630/650, which can be conveniently coupledto primary amines using an active ester derivative that is availablecommercially (Molecular Probes, Inc., Catalog number, B-10003); (ii)BODIPY 650/665, which can be conveniently coupled to primary aminesusing an active ester derivative that is available commercially(Molecular Probes, Inc., Catalog number, B-10005); (iii) Dansyl, whichcan be conveniently coupled to primary amines using dansyl chloride thatis commercially available (Aldrich Chemical); (iv) Rhodamine, which canbe conveniently coupled to primary amines using an active esterderivative that is available commercially (Molecular Probes, Inc.,Catalog number, R-6107); (v) 5-TAMRA, which can be conveniently coupledto primary amines using an active ester derivative that is availablecommercially (Molecular Probes, Inc., Catalog number, C-2211).

As noted, in one aspect of the present invention, preferred fluorophoresinclude macrocyclic fluorescent dye compounds, especially compoundshaving aromatic π-electron systems. These dye compounds act asmultidentate macrocyclic ligands to chelate a central complexing atom.Thus, these preferred fluorophore moieties may comprise a substantiallyplanar multidentate macrocyclic ligand coordinated to a complexingcentral ion or atom. Preferred elements include aluminum, phosphorous,and the group IVB elements, e.g. silicon, germanium, and tin.

Other suitable fluorophores include coumarin dyes, nitrobenzoxazoledyes, cyanine dyes, dipyrrometheneboron dyes, xanthene dyes (includingthe benzo- and naphtho-xanthene dyes), phenoxazine dyes (as well as thebenzo- and naphtho-phenoxazine dyes) and compounds from other classes ofdyes well known to those of skill in the art. Other suitablefluorophores include the fluorophores in the following non-exclusivelist: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethyhhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Amninoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red;Acridine Yellow;Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AutoFluorescent Protein—(QuantumBiotechnologies) see sgGFP, sgBFP; Alexa Fluor (various)™; AlizarinComplexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCAAminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aminomethylcoumarin (AMCA); Anilin Blue 600; Anthrocyl stearate; APC(Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn2+; APTS; AstrazonBrilliant Red 4G, Orange R, Red 6B, and Yellow 7 GLL; Atabrine;ATTO-TAG™ CBQCA and FQ; Auramine; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high or low pH); Berberine Sulphate;Beta Lactamase; BFP blue shifted GFP; Blue Fluorescent Protein; blueshifted GFP (Y66H); BFP/GFP FRET; Bimane; Bisbenzamide; bis-BTC=RatioDye, Zn2+; Blancophor FFG and SV; BOBO™-1 and -3; Bodipy (various);BO-PROTM-1 and -3; Brilliant Sulphoflavin; BTC—Ratio Dye Ca2+; BTC-5N;Calcein; Calcein Blue; Calcium Crimson™; Calcium Green (various) andOrange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blues™;Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP=13 CyanFluorescent Protein; Cyan Fluorescent Protein; CFP/YFP FRET;Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA;Coelenterazine Dye (various); Coumarin Phalloidin; C-phycocyanine;Methylcoumarin; Methylcoumarin CTC; CTC Formazan; Cy2™ and variousothers; Cyan GFP; cyclic AMP Fluorosensor (FiCRbR); Dabeyl (various);Dansyl (various); DAPI; Dapoxyl (various); DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS; DiA (4Di-16-ASP); DichlorodihydrofluoresceinDiacetate (DCFH); DiD—Lipophilic Tracer, DiD (DiIC18(5)); DIDS;Dihydorhodamine 123 (DHR); DiI (IilC18(3)); Dinitrophenol; DiO(DiOC18(3)); DiR (various); DM-NERF (high pH); DNP; Dopamine; DsRed;DTAF; DY-630-NHS; DY-635-NHS; EBFP; Enhanced Blue Fluorescent Protein;Enhanced Cyan Fluorescent Protein; Enhanced Green-Fluorescent Protein;ELF; Eosin; Erytbrosin; Erythrosin; Ethidium Bromide; Ethidiumhomodiiner-1 (EthD-1); Euchrysin; EukoLight; Europium (II) chloride;EYFP; Enhanced Yellow Fluorescent Protein; Fast Blue; FDA; Feulgen(Pararosaniline); FIF (Formaldehyd Induced Fluorescence); Flazo Orange;Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby, FluorX;FM 1-43™; FM 4-46; Fura Red™ (various); Genacryl Brilliant Red B,Brilliant Yellow 10GF, Pink 3G, and Yellow 5GF; GeneBlazer (CCF2); GFP(various); Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst33258, 33342, and 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indo-1 (various); Indodicarbocyanine(DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1;JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF, SF, and WS;Lissamine Rhodarnine (various); Calcein/Ethidium homodimer; LOLO-1;LO-PRO-1; Lucifer Yellow; Lyso Tracker (various); LysoSensor (various);Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2;Mag-Fura-5; Mag-Indo-1; Magnesium Green and Orange; Malachite Green;Marina Blue; Maxilon Brilliant Flavin; Maxilon Brilliant Flavin;Merocyanin; Methoxycoumarin; Mitotracker Green, Orange, and Red;Mitramycin; Monobromobimane; Monobromobirmane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant lavin; Oregon Green™ (various); Pacific Blue;Pararosaniline (Feulgen); PBFI; PE-Cy5 and Cy7; PerCP; PE-TexasRed [Red613]; Phloxin B (Magdala Red); Phorwite (various); Phosphine;PhotoResist; Phycoerythrin B and R; PKH26 and 67; PMIA; Pontochrome BlueBlack; POPO-1 and -3; PO-PRO-1 and -3; Primuline; Procion Yellow;Propidium Iodid (PI); PyMPO; Pyrene; Pyronine; Pyronine B; PyrozalBrilliant Flavin; QSY; Quinacrine Mustard; Red 613 [PE-TexasRed];Resorufin; Rhodamine (various); Rose Bengal; R-phycocyanine;R-phycoerythrin (PE); red shifted GFP (various); Sapphire GFP; SBFI;Serotonin; Sevron Brilliant Red, Orange, and Yellow L; SuperGlo™GFP(various); SITS (various); SNAFL (various); SNARF; Sodium Green; Spectrmqua, Green, Orange, and Red; SPQ(6-methoxy-N-(3-sulfopropyl)quiolinium); Stilbene; Sulphorhodamine B andG; SYTO (various); SYTOX Blue, Green, and Orange; Tetracycline;Tetramethylrhodanine (TRITC); Texas Red™; Texas Red-X™ conjugate;Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin5, S, and TCN; Thiolyte; Thiozole Orange; Tinopol CBS (CalcofluorWhite); TMR; TO-PRO-1, -3 and -5; TOTO-1 and -3; TriColor (PE-CyS);TRITC; TetramnethylRodamineIsoThioCyanate; True Blue; TruRed; Ultralite;Uranine B; Uvitex SFC; X-Rhodamine; XRITC; Xylene Orange; Y66F, H, andW; Yellow shifted Green Fluorescent Protein; Yellow Fluorescent Protein;YO-PRO-1 and -3; and; YOYO-1 and -3.

Linking Groups

The fluorophores used herein can be attached to the glucose ordeoxyglucose or glucose or deoxyglucose derivative using any linkagechemistry that is compatible with the fluorophore and the glucose ordeoxyglucose or glucose or deoxyglucose derivative portions of theconjugate.

In some embodiments, the fluorophore can be attached directly to glucoseor deoxyglucose or a glucose or deoxyglucose derivative using, forexample, a fluorophore isothiocyanate (e.g., fluorescein isothiocyanate)and a suitably protected glucose or glucose derivative (e.g., tetraO-acetyl-2-deoxy-2-aminoglucose) to form a desired conjugate. A numberof protected glucose or deoxyglucose compounds or derivatives arecommercially available or can be prepared according to well-establishedmethods. In many instances, the glucose or deoxyglucose moiety having“n” hydroxy or amino groups, will have “n-1” protecting groups, therebyleaving one available reactive functional group as an attachment sitefor either the fluorophore or a linking group. Additionally, theunprotected functional group will be at a known location on the glucose,deoxyglucose or derivative thereof to prepare conjugates of a desiredstructure.

In certain embodiments, a linking group is used to attach thefluorophore to the glucose or deoxyglucose or glucose or deoxyglucosederivative covalently. In these embodiments, any commercially availablebifunctional linking groups, preferably, heterobifunctional linkinggroups (see Pierce Catalog), can be used. Alternatively, one of skill inthe art can construct a linking group having two or more reactivefunctional groups to attach the fluorophore and the glucose ordeoxyglucose moiety (e.g., heterobifunctional or homobifunctional). Insome embodiments, either a plurality of glucose or deoxyglucose moietiesor a plurality of fluorophores or both are attached to a multifunctionallinking group to provide a conjugate of the invention.

The terms “linker” and “linking group” refer to a moiety that is used toconnect various portions of the conjugate to one another. Typically alinker or linking group has functional groups that are used to interactwith and form covalent bonds with functional groups in the components(e.g., fluorophores and glucose or glucose derivatives) of theconjugates described and used herein. Examples of functional groups onthe linking groups (prior to interaction with other components) include—NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —OH, —CO₂H or —H. One of skill inthe art will understand that each of these functional groups can form acovalent linkage to a suitable functional group on the glucose portionor the fluorophore portion of the conjugate during synthesis of theconjugate. For example, amino, hydroxy and hydrazino groups can eachform a covalent bond with a reactive carboxyl group (e.g., a carboxylicacid chloride or activated ester such as an N-hydroxysuccinimide ester(NHS)). Other suitable bond forming groups are well-known in theliterature and can be used to prepare conjugates of the presentinvention.

The linking group can include linear or acyclic portions, cyclicportions, aromatic rings or combinations thereof More specifically, thelinking group L will typically have from 3 to 100 main chain atoms otherthan hydrogen atoms, selected from C, N, O, S, P and Si, and will becyclic, acyclic, aromatic or a combination thereof. Additionally, thelinking groups will be sufficiently robust so that they are stable toreaction conditions used in conjugate assembly, e.g., theprotection/deprotection chemistries used to prepare the conjugates ofthe invention. Illustrative linkers and synthetic chemistries aredescribed in more detail below.

In addition to commercially available linking groups, U.S. Pat. Nos.5,512,667; 5,451,463; and 5,141,813 describe other linking groups thatcan be used to prepare conjugates of the present invention. U.S. Pat.Nos. 5,696,251; 5,585,422; and 6,031,091 describe certaintetrafunctional linking groups that can be useful in preparingconjugates of the present invention, such as, for example, conjugatescomprising two or more fluorophores. Functional groups or linkers usefulin preparing conjugates of the present invention include primary andsecondary nitrogen, primary and secondary OH, and —SH.

As discussed in the following section, a preferred deoxyglucose moietyfor forming the conjugates of the present invention is D-glucosamine asthe hydrochloride salt. This compound has an amino group that provides achemical handle for conjugations. Fluorophores can be conjugateddirectly to this amino group via alkyl, amide, or sulfonamide linkages,for example. Alternatively, a linker or spacer can separate thefluorophore and the glucosamine. The glucosamine is then not stericallyimpacted by the fluorophore and is free to act like glucose in term ofuptake via the glucose transport system and phosphorylation via thehexokinase enzymes. Optimal linkers are oligomers of ethylene glycol orstraight alkyl chains. These linkers are attached to the glucosamineamine via either an alkyl or amide connection. The fluorophore isattached to the other end via an amide, sulfonamide, or etherconnection. The optimum length of the linker is from 4 to 16 atoms.Illustrative synthetic schemes for forming such conjugates of theinvention are shown below for several preferred linkers of theinvention. The 5-(and 6) carboxynaphtofluorescein is shown as an exampleof a fluorophore (only one isomer shown in schemes), but any fluorophoreas an N-hydroxysuccinide (NHS) ester can be used in these illustrativeschemes to form a conjugate of the invention.

Synthesis of carboxynaphtofluorescin-oligo-PEG amido-glucosamineconjugates

The length of the linker arm can be varied. For example, in the case ofthe amide connections, one can use the derivatives shown below in placeof N-t-boc-amido-PEG4-acid, product number 10220, Quantabiodesign Inc.

These t-boc protected amino acids can be derived from the correspondingamino acid and BOC-ON (19,337-2, Aldrich Chemical). The amino acids inturn can be obtained from published procedures in the chemicalliterature.

Synthesis of carboxynaphtofluorescin-oligo-PEG-alkyl-glucosamineconjugate

The length of the inker arm can be varied. For example, in the case ofthe alkyl connection, one can use the derivatives shown below in placeof N-t-boc-amido-PEG4-alcohol, (product number 10250, QuantabiodesignInc.).

These t-boc protected amino alcohols can be derived, for example, fromthe corresponding amino alcohols and BOC-ON (19,337-2, AldrichChemical). The amino alcohols in turn can be obtained from publishedprocedures in the chemical literature.

Alternatively, the starting amino acids and alcohols for the conjugatescan comprise all alkyl chains as shown below. The amino acids andalcohols are available commercially (Aldrich Chemical) and can bederivatized with BOC-ON (19,337-2, Aldrich Chemical) to produce thereagents shown below.

Glucose and Deoxyglucose and Their Derivatives

The glucose, deoxyglucose or derivative components of the conjugatesdescribed herein are preferably D-(+)-deoxyglucose, D-(+)-glucosamine,and N-acetyl D-glucosamine. Other monosaccharides (e.g., 5-thio-glucose,D-galactose and D-fructose) and their derivatives can also be used. Inaddition, any of the monosaccharides and their derivatives described inPCT patent publication WO 01/82926, incorporated herein by reference,can be used to prepare a conjugate of the invention.

Some compounds are preferred for the conjugates used in the presentmethods. In other work,6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG)was used to study glucose uptake in certain cells. See, for example,Yoshioka et al., 1996, Biochimica et Biophysica Acta 1289: 5-9; Yamadaet al., 21 Jul. 2000, J. Biol. Chem. 275(29): 22278-22283; Ball et al.,2002, Can. J. Physiol. Pharmacol. 80: 205-209; and Leira et al., 2002,Toxicology in Vitro 16: 267-273, each of which is incorporated herein byreference. This particular deoxyglucose conjugate is labeled with NBD atthe 6-carbon position. The hydroxy group at the 6-carbon position istypically phosphorylated upon entry into a cell, inhibiting itsdiffusion from the cell. With conjugates carrying a fluorophore at thisposition, phosphorylation is less likely to occur, and the conjugatewill thus be more likely to diffuse out of the cells. Conversely,fluorescent labeling of glucose or deoxyglucose at the 2-position or3-position results in conjugates that can be phosphorylated after uptakeby a cell, with the consequence that such conjugates are less likely todiffuse out of the cells. In addition, deoxyglucose derivatives areoften preferred, as they are less likely to be hydrolyzed by esters andother enzymes in the cell. By accumulating the conjugate in the cells,sensitivity of the present method is increased; thus, conjugates of thepresent invention that have the fluorophore linked to the 2- or3-position of the glucose, deoxyglucose or derivative are preferred.

Accordingly, in one embodiment, the fluorophore glucose, deoxyglucose or20 derivative conjugate of the present invention has a formula selectedfrom:

wherein Fl and L have the meanings provided above.

Another preferred embodiment of the fluorophore glucose, deoxyglucose or25 derivative conjugate of the invention has the formula:

wherein Fl is a fluorophore; L is a linking group; each X isindependently selected from the group consisting of O and NH; and R¹, R³and R⁴ are each members independently selected from the group consistingof H, (C₁-C₁₂)alkyl, (C₁-C₁₂)acyl and a solubility or partitioningeffector component. The solubility or partitioning effector component isa component that increases the solubility of the resultant conjugate inaqueous solution relative to a conjugate having a hydrogen atom at thesame position. Suitable solubility or partitioning effector componentsinclude oligoethylene glycol, oligopropylene glycol, polyhydroxylatedcarbon chains (typically two to thirty carbons in length) and the like.

In a preferred embodiment, the fluorophore glucose, deoxyglucose orderivative conjugate has the formula Ic in which each X is O, and R¹, R³and R⁴ are each H.

In another preferred embodiment, the fluorophore glucose, deoxyglucoseor derivative conjugate has the formula Ic in which each X is O, and twoof R¹, R³ and R⁴ are H, with the remaining member of R¹, R³ and R⁴ beinga solubility or partitioning effector component.

In a most preferred embodiment, L is attached to a glucose ordeoxyglucose derivative by means of an amino or amido linkage (e.g., theglucose derivative is a 2-amino-2-deoxyglucose derivative).

Administering the Fluorophore Glucose or Deoxyglucose Conjugate

Administration of a fluorophore glucose or deoxyglucose conjugateprovided herein can be effected by any method that enables delivery ofthe conjugates to the site of the cancer or suspected cancer. In oneembodiment, delivery is via circulation in the bloodstream. To place theconjugates in contact with cancerous tissues or cells, the methods ofadministration include oral, buccal intraduodenal, parenteral injection(including intravenous, subcutaneous, intramuscular, intravascular, orinfusion), topical administration, and rectal.

The amount of the conjugate administered will be dependent upon thesubject being treated, the severity of the cancer, localization of thecancer, the rate of administration, the disposition of the conjugate(e.g., solubility and fluorescence intensity) and the discretion of theadministrator. However, an effective dosage is typically in the range ofabout 0.001 to about 100 mg per kg body weight, preferably about 1 toabout 35 mg/kg/day, in single or divided doses. For a 70 kg human, thisdosage would amount to about 0.05 to about 7 g/day, preferably about 0.2to about 2.5 g/day. In some instances, dosage levels below the lowerlimit of the aforesaid range may be more than adequate, while in othercases still larger doses may be employed without causing any harmfulside effect, although such larger doses may be divided into severalsmaller doses for administration throughout the day.

The imaging fluorophore conjugate composition may, for example, be in aform suitable for oral administration, such as a tablet, capsule, pill,powder, sustained release formulation, solution, or suspension; forparenteral injection, such as a sterile solution, suspension oremulsion; for topical administration, such as an ointment or cream; orfor rectal administration, such as a suppository. The imagingfluorophore conjugate composition may be in unit dosage forms suitablefor single administration of precise dosages and can include aconventional pharmaceutical carrier or excipient.

Exemplary parenteral administration forms include solutions orsuspensions of the imaging conjugate in sterile aqueous solutions, forexample, aqueous propylene glycol or dextrose solutions. Such dosageforms can be suitably buffered, if desired.

Suitable pharmaceutical carriers include inert diluents or fillers,water, and various organic solvents. The pharmaceutical compositionsmay, if desired, contain additional ingredients such as flavorings,binders, excipients, and the like. Thus for oral administration, tabletscontaining various excipients, such as citric acid, may be employedtogether with various disintegrants such as starch, alginic acid, andcertain complex silicates, and with binding agents such as sucrose,gelatin, and acacia. Additionally, lubricating agents such as magnesiumstearate, sodium lauryl sulfate, and talc are often useful for tabletingpurposes. Solid compositions of a similar type may also be employed insoft and hard filled gelatin capsules. Preferred materials, therefore,include lactose or milk sugar and high molecular weight polyethyleneglycols. When aqueous suspensions or elixirs are desired for oraladministration the imaging fluorophore conjugate therein may be combinedwith various sweetening or flavoring agents, coloring matters or dyes,and, if desired, emulsifying agents or suspending agents, together withdiluents such as water, ethanol, propylene glycol, glycerin, orcombinations thereof.

Methods of preparing various pharmaceutical compositions with a specificamount of an active ingredient that are suitable for use with the activeimaging fluorophore conjugates of the present invention are known, orwill be apparent upon consideration of the disclosure herein, to thoseskilled in this art. For examples, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).

Because the imaging fluorescent conjugates of the present invention arepreferentially taken up by cancer cells, it is possible to obtain animage of or visually confirm the presence of cancer cells that havetaken up the conjugate. Detection of the conjugates can be performedusing essentially any fluorescence detection device to obtain an imageof the cancerous tissues or cells.

Detection of Cancer

Detection and imaging of tissues or cells that take up the conjugatesdescribed herein can be accomplished using visual techniques or viatwo-dimensional image information processing by direct continuousobservation with a fluorescence microscope. While spatial resolution canbe difficult for certain visual methods (unaided by spectral enhancersor microscopes), a typical fluorescence microscope can providesufficient resolution at a single cell level.

For example, with a confocal laser scanning fluorescent microscope,3-dimensional stereoscopic image information with a resolution of about1 μm can be continuously obtained in real time from tissues in vivo. Avariety of known methods can be adapted for use with the conjugates ofthe present invention. For example, the conjugates of the invention canbe used in the endoscopic technique described in U.S. Pat. No.5,261,410, in which an infrared monochromatic light source is employedand the Raman shift in emission radiation is measured to assess thetissue. Likewise, PCT patent publication No. WO 96/10363 discloses amethod of normalization by dividing the intensity at each wavelength bythe integrated area under the spectrum. Differences in the resultingcurves are then used as the basis for diagnosis.

One of skill in the art will appreciate that essentially anyfluorescence detection means, either microscopic or macroscopic, can beemployed that is capable of detecting the fluorophore glucose ordeoxyglucose conjugate localized in a particular lesion, tissue, organ,or cell.

In some embodiments, the detection means can be in the form of anendoscope inserted into a body cavity through an orifice, such as themouth, nose, ear, anus, urethra, vagina or an incision. The term“endoscope” is used here to refer to any scope introduced into a bodycavity, e.g., an anally introduced endoscope, an orally introducedbronchoscope, a urethrally introduced cystoscope, an abdominallyintroduced laparoscope, and the like. The miniaturization of scopecomponents has greatly enhanced the utility of an endoscope, makingendoscopes particularly useful in the practice of the present invention.

In addition to methods of detecting cancer as generally described above,certain embodiments of the present invention relate to intraoperative,laparoscopic, intravascular, and endoscopic examination, biopsy andtreatment of tissues and/or organs with a fluorophore conjugatedetecting means capable of close approach to suspected sites of tumorrecurrence, metastasis, or incomplete removal of cancer tissue. As usedherein, endoscopic procedures include laparoscopic procedures.

Embodiments of the present invention also relate to the intravascular,intraoperative, laparoscopic, and endoscopic examination of lesions witha fluorophore conjugate detecting means capable of close approach tosuspected sites of the lesions, especially non-malignant pathologicallesions. Lesions include cancerous, hyperplasic, and pre-cancerous cellsor tissues.

As noted above, the methods of the present invention do not requireprocessing of images. Rather, in one embodiment, a surgeon or clinician,through the use of, e.g., an intraoperative, laparoscopic, intravascularprobe or an endoscope, can quickly scan areas of suspected tumor growthand use the level of fluorescence to more precisely discriminate tumortissue from non-tumor tissue and thereby more precisely define tumorborders for surgical resection or diagnostic evaluation, or for laser orradiation therapy, including brachytherapy and external beam therapy, orfor improved biopsy procedures.

Other embodiments enable the intravascular, intraoperative,laparoscopic, or endoscopic detection means to be similarly used todefine and treat lesions. In another embodiment, the conjugate is usefulfor therapy of the detected tumor by emitting oxygen free radicals orother byproducts which damage the cells in which there has beenaccumulation of the conjugate. The emission of such damaging agents canbe aided or induced by the energy which excites the fluorophore.

The above detection methods can be carried out in combination with asurgical procedure, such as a cancer resection. The method of detectingcan be carried out endoscopically, for example, or visually as part of askin examination for melanoma screening.

Methods of Detecting or Imaging Pre-Cancer

In a related aspect, the present invention provides methods fordetecting pre-cancerous cells in a subject, comprising:

(a) administering to the subject an effective amount of a fluorophoreglucose, deoxyglucose or derivative conjugate; and

(b) detecting cells that take up the fluorophore glucose or deoxyglucoseconjugate to determine if pre-cancerous cells are present in thesubject.

The fluorophore conjugate used in this aspect of the invention ispreferably one having the formula provided above as (I); more preferablyhaving the formula provided above as (Ia) or (Ib); and most preferablyone having the formula (Ic).

The application of the present method to detection and imaging ofpre-cancerous cells (e.g., dysplastic cells) resides in the upregulatedglucose transport that is exhibited by pre-cancerous cells. Generally,the conjugates, methods of administration and detection are the same ashave been described above with respect to the detection and imaging ofcancer cells.

Methods for Cancer or Pre-Cancer Detection During an Operative orEndoscopic Procedure

As noted above, the detection methods provided herein are broadlyapplicable and can be particularly effective when used in combinationwith surgical or endoscopic procedures.

Accordingly, in another related aspect, the present invention provides amethod for cancer or pre-cancer detection during an operative orendoscopic procedure, the method comprising:

(a) administering to a patient subject to said procedure an effectiveamount of a fluorescent glucose, deoxyglucose or derivative conjugate,the conjugate having a rate of uptake in cancerous or pre-cancerouscells that is at least two times greater than the rate of uptake innormal cells;

(b) conducting the procedure within 48 hours of administering theconjugate; and

(c) scanning the patient with a detection means for detecting thelocalization of the fluorescent conjugate in cancerous or pre-cancerouscells.

In this aspect, a fluorescent conjugate is administered to a patientprior to or coincident with a surgical procedure for the removal ofcancerous tissue. The conjugate can be administered by any methoddesigned to bring the conjugate into close proximity with the canceroustissue, tumor, or lesion to be removed. In this manner, uptake of theconjugate by cancer cells will provide a more accurate determination ofthe cancer margins.

To facilitate detection during the operative or endoscopic procedure,the conjugate will typically be one that exhibits a rate of uptake incancerous or precancerous cells that is at least two times greater thanthe rate of uptake in normal cells (e.g., PBMC cells). Evaluation ofsuch uptake can be carried out as described in the Examples below.Preferably, the rate of uptake in cancerous or pre-cancerous cells is atleast 10 times the rate of uptake in normal cells, more preferably atleast 20 times, and still more preferably at least 40 times.

For those embodiments in which the fluorescent conjugate is administeredprior to the operative or endoscopic procedure, it will preferably beadministered from about 1 hour to about 48 hours prior to the procedure,more preferably from about 1 hour to about 16 hours prior to theprocedure, and most preferably from about 1 hour to about 6 hours priorto the procedure.

During the procedure, and depending upon the conjugate used, detectioncan be visual. In some embodiments, the fluorescence of cells that havetaken up the conjugate can be enhanced by excitation of the fluorophorewith light of a suitable wavelength. Accordingly, once a portion of thetumor or lesion is removed, the remaining tissue can be subjected to asuitable light source to excite the fluorescent conjugates that remainand additional resection can be accomplished.

In other embodiments, detection can be accomplished using fluoroscopesand other detection devices known to those of skill in the art.

Fluorophore Glucose, Deoxyglucose and Derivative Conjugates

In yet another aspect, the present invention provides fluorophoreglucose, deoxyglucose and derivative conjugates having the formula:Fl-L-Glcwherein Fl is a fluorophore having an emission wavelength of from about400 nm to about 1200 nm; L is a bond or a linking group; and Glc isglucose, deoxyglucose or derivative, including deoxyglucose andderivatives thereof, with the proviso that the conjugate is other than2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose.

As with the methods above, this aspect of the invention is directed to avariety of glucose, deoxyglucose and derivative conjugates. Inparticular, the fluorophores used in this aspect of the invention willtypically be selected from coumarin dyes, nitrobenzoxazole dyes, cyaninedyes, dipyrrometheneboron dyes, xanthene dyes (including the benzo- andnaphtho-xanthene dyes), phenoxazine dyes (as well as the benzo- andnaphtho-phenoxazine dyes) and other classes well known to those of skillin the art. Illustrative embodiments of the fluorophore glucose ordeoxyglucose conjugates of the invention and methods for theirpreparation are provided in the Examples below.

In one group of embodiments, L is a bond such that the fluorophore Fl isdirectly attached to Glc. Typically, this attachment is accomplished viacoupling of a functional group on Fl with a compatible (e.g.,linkage-forming) functional group on Glc. In certain preferredembodiments, Fl has an isocyanate, isothiocyanate or carboxylic acidfunctional group that is used to attach Fl to a hydroxy or amino grouppresent on Glc to form a carbamate, thiocarbamate, urea or thiourealinkage between the components.

In another group of embodiments, L is a-linking group which can beessentially any of the linking groups discussed above and generallyknown to those of skill in the art. The linking group in this aspect ofthe invention typically has from 3 to 100 main chain atoms other thanhydrogen atoms, selected from C, N, O, S, P and Si, and can be cyclic,acyclic, aromatic or a combination thereof. Additionally, the linkinggroups are sufficiently robust so that they are stable not only toreaction conditions used in conjugate assembly, e.g., theprotection/deprotection chemistries used to prepare the conjugates, butare also stable to conditions within a cancer cell. More particularly,the linking group is one that is stable to hydrolytic and proteolyticconditions that are typically found in a cancer cell (e.g., exhibits at_(1/2) for hydrolysis or cleavage of the linkage of at least about 4hours at ambient temperatures in a cancer cellular milieu). Preferredlinking groups include both homo- and hetero-bifunctional linkinggroups, as are commercially available or readily prepared according toknown methods.

The glucose or glucose derivative components of the conjugates describedherein are preferably D-(+)-deoxyglucose, D-(+)-glucosamine and N-acetylD-glucosamine. Other monosaccharides (e.g., D-galactose and D-fructose)can also be used.

In one group of preferred embodiments, the fluorophore glucose ordeoxyglucose conjugate has a formula selected from:

wherein Fl and L have the meanings provided above.

Still other preferred embodiments are those fluorophore glucose,deoxyglucose or derivative conjugates wherein the glucose portion is a2-deoxy glucose, the conjugate having the formula:

wherein Fl is a fluorophore; L is a linking group; each X isindependently selected from the group consisting of O and NH; R¹, R³ andR⁴ are each members independently selected from the group consisting ofH, (C₁-C₁₂)alkyl, (C₁-C₁₂)acyl and a solubility or partitioning effectorcomponent. The solubility or partitioning effector component can beessentially any component that increases the solubility of the resultantconjugate in aqueous solution relative to the conjugate having ahydrogen atom at the same position. Suitable solubility or partitioningeffector components include oligoethylene glycol, oligopropylene glycol,polyhydroxylated carbon chains (typically two to thirty carbons inlength) and the like.

In a most preferred embodiment, the fluorophore conjugate has theformula Ic in which each X is O, and R¹, R³ and R⁴ are each H.

In another most preferred embodiment, the fluorophore conjugate has theformula Ic in which each X is O, and two of R¹, R³ and R⁴ are H, withthe remaining member of R¹, R³ and R⁴ being a solubility or partitioningeffector component.

In the most preferred embodiment, L is attached to the glucose frameworkby means of an amino or amido linkage (e.g., the deoxyglucose is a2-amino-2-deoxyglucose).

Kits for Cancer Detection

In still another aspect, the present invention provides kits for use bya clinician, the kits having instructions for administration and cancerdetection along with a sterile preparation of a fluorophore conjugateand a pharmaceutically acceptable carrier.

Other Applications

The fluorophore conjugates of the invention, while highly useful for thedetection of cancer cells and tissues, can also be used in anyapplication where a fluorescently labeled glucose, deoxyglucose orderivative molecule is useful. In one embodiment, the compounds of theinvention are used to measure glucose uptake. In another embodiment, thecompounds of the invention are used in an assay to determine whether acompound binds glucose or deoxyglucose. Likewise, while the methods andconjugates of the invention are especially valuable for the diagnosisand detection of cancer in humans, the methods and conjugates of theinvention will also find application in the study and treatment ofcancer in animals.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1

This example illustrates the time course of uptake of 2-NBDG in Rajilymphoma cells.

Materials:

Raji cells from culture of one t-75 flask. Phosphate-buffered salineplus 2.5 mM CaCl₂, 1.2 mM MgSO₄, 4 mM KCl and 5 mM glucose (PBS+).2-NBDG can be prepared in accordance with the method reported inYoshioka et al., 1996, Biochimica et Biophysica Acta 1289: 5-9,incorporated herein by reference.

Uptake Reaction:

Culture medium with cells (15 mL) was centrifuged for 10 min at 800×g.The pellet of cells was resuspended in PBS+ and centrifuged for 10 minat 800×g, and the resulting pellet was resuspended in 1.5 mL PBS+.Aliquots (100 uL) were placed into each of 8 tubes and incubated at 37°C. for 15 min. The fluorophore deoxyglucose conjugate 2-NBDG was addedto each tube to provide a concentration of 200 uM. A tube was removedfrom the 37° C. bath at each of 0, 1, 3, 5, 10, 20, 30 and 40 min andimmediately placed on ice. The cells were washed twice with PBS andresuspended in 100 uL of PBS. A 20 uL aliquot was placed in each of 5wells of an LJL 96-well plate and fluorescence was counted by a LJLplate reader. FIG. 1 shows the results of the time course experiment.

EXAMPLE 2

This example illustrates a comparison of 2-NBDG uptake in Raji lymphomacells and peripheral blood white cells and illustrates the preferreduptake of a fluorophore deoxyglucose conjugate by a cancer cell ascompared to a normal cell.

Materials:

Raji cells from culture one t-75 flask. Peripheral blood white cellsfrom Stanford Blood Bank. Leukocytes were further separated by Ficollseparation (twice). These cells are referred to as PBMC peripheral bloodmononuclear cells). Phosphate-buffered saline. 2-NBDG, 0.1 M in water.

Uptake Reaction:

Culture medium with cells (either Raji or PBMC, 15 mL) were centrifugedfor 10 min at 800×g. The pellet of cells was resuspended in PBS andcentrifuged for 10 min at 800×g, and the resulting pellet wasresuspended in 1.5 mL PBS. Cells were counted and adjusted to 1×10⁶ permL for both. Aliquots (100 uL) were placed into each of 2 tubes for eachcell type, and the tubes were incubated at 37° C. for 15 min. Thefluorescent deoxyglucose conjugate 2-NBDG was added to each tube toprovide a concentration of 200 uM. At 40 min, the tubes were removedfrom the 37° C. bath and immediately placed on ice. The cells werewashed twice with PBS and resuspended in 100 uL of PBS. A 20 uL aliquotwas placed in each of 5 wells of an LJL 96-well plate, and fluorescencewas counted by a LJL plate reader. FIG. 2 shows the results of the timecourse experiment.

EXAMPLE 3

This example illustrates the preparation of the fluorophore deoxyglucoseconjugate of the invention FGC-002. In the conjugate, the fluorophore isBODIPY, and the glucose derivative is glucosamine.

Generally, the FGC-002 conjugate is prepared by treating6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid, succinimidyl ester (BODIPY from Molecular Probes, D-2184, MW 502)with an excess of D-glucosamine (Sigma) in an aprotic solvent withgentle heating. Isolation of the product (FGC-002) can be accomplishedvia chromatography.

More particularly, 2.2 mg (4 micromoles) of6-((4,4-difluoro-5,7-diethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid, succinimidyl ester was dissolved in 0.3 ml of DMF followed by theaddition of 6 mg (28 micromoles) of glucosamine HCL dissolved in 0.3 mlof water and 3.9 microliters (28 micromoles) of triethyl amine. Thereaction was stirred for 24 hrs at room temperature, evaporated underreduced pressure, and dissolved in a minimum of methanol. This solutionwas spread on a preparative TLC plate (silica), dried, and developedwith 2.5% water in acetonitrile. The plate was then dried and theproduct visualized with a UV light. The only fluorescent band wasscraped off the plate and then eluted with 10% water in acetonitrile andevaporated under reduced pressure, yielding 1 mg of product, which wassoluble in methanol and sparingly soluble in water.

FIG. 3 illustrates a time-course uptake of FGC-002 relative to2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in Rajilymphoma cells. The uptake study was conducted substantially inaccordance with the protocol described in Example 1.

EXAMPLE 4

This example provides synthetic methods for other illustrativefluorophore deoxyglucose conjugates of the invention.A. Synthesis of Texas Red Glucosamine Conjugate

About 4 mg (4.8 micromoles) of Texas Red®-X, succinimidyl ester *singleisomer* (816.94 MW, Molecular Probes, T-20175) were dissolved in 1.2 mlof methanol and combined with 4 mg (18 micromoles) of glucosamine HCl(Sigma) in 600 microliters of water. About 2.5 microliters (18micromoles) of triethylamine were then added. The reaction was stirredovernight at 20° C. and then evaporated under reduced pressure. The gumwas dissolved in methanol and spread on a preparative TLC plate(silica), dried, and developed with 10% water in acetonitrile. The platewas then dried and the product visualized with a UV light. The only redfluorescent band was scraped off the plate and then eluted with 20%water in acetonitrile and evaporated under reduced pressure, yielding 2mgs of product which was soluble in methanol and sparingly soluble inwater.B. Synthesis of 5-(and 6) Carboxynaphtofluorescein Glucosamine Conjugate

About 8.3 mg (14 micromoles) of 5-(and-6)-carboxynaphthofluorescein,succinimidyl ester *mixed isomers* (MW 573.51, Molecular Probes, C-653)were dissolved in 2 ml of DMF, and 10 mg (46 micromoles) of glucosamineHCl (Sigma) in 1 ml of water was added. About 5.8 microliters (46micromoles) of triethylamine were then added. The reaction was stirredovernight at 20° C. and then evaporated under reduced pressure. The gumwas dissolved in 25% water in methanol and spread on a preparative TLCplate (silica), dried, and developed with 10% water in acetonitrile. Theplate was then dried and the product visualized with a UV light. Theonly fluorescent band was scraped off the plate and then eluted with 20%water in acetonitrile and evaporated under reduced pressure, yielding 2mgs of product, which was soluble in methanol and sparingly soluble inwater.C. Synthesis of Glucosamine N-methylanthranilamide Conjugate

To a slurry of glucosamine (0.25 g, 1.2 mmol) and NEt₃ (1.0 mL, 7.0mmol) in DMF (1 mL) was added a solution of N-methylitacoicanhydride(0.1 g, 0.56 mmol), and the resulting mixture was stirred at 80° C. for18 h. The reaction mixture was filtered, adsorbed upon silica gel andseparated on a silica gel column by washing with acetonitrile andeluting with 10% water in acetonitrile to yield 5 mg of the desiredproduct.

EXAMPLE 5

This example illustrates the application of fluorophore conjugates ofthe present invention to procedures such as gastrointestinalcolonoscopy.

Gastrointestinal Colonoscopy:

Endoscopic evaluation of the gastrointestinal tract is recommended forall individuals older than 50 years of age. This screening examinationhelps the physician identify polyps or other lesions which may beprecancerous or cancerous but still localized in the intestine.Identification and removal of these lesions prevent spread of thedisease and can cure most patients. This procedure suffers from somesignificant limitations, including difficulty in distinguishing betweenhyperplastic lesions and adenomatous lesions, the latter of which mustbe removed. With knowledge that a polyp is hyperplastic and notcancerous, the lesion can be ignored without removal and biopsy. Inaddition, some lesions are small and flat. These lesions are frequentlyoverlooked and can be or become cancerous. Due to current limitations,some lesions are incompletely removed during the endoscopic procedure,which leads to an increased risk of recurrence.

To eliminate or ameliorate these limitations of endoscopy, fluorophoreconjugates are administered to the patient by intravenous injection from1 to 48 hours, preferably 2 to 6 hours, prior to the endoscopicprocedure. The conjugate can be administered as an oral solution or bypill or as a suppository. The fluorophore conjugate circulates in theblood and accumulates in cancerous or precancerous tissue relative tonormal or hyperplastic tissue. During endoscopy, the physician can viewthe colon under both white light and fluorescent capture. Specifically,while using white light, the physician sees a lesion that appears to beeither hyperplastic or adenomatous. In particular, these lesions areviewed with fluorescent capture to determine if the lesion must beresected or can be ignored. Lesions that are highly fluorescent areclearly malignant or premalignant and are removed. Prior to completingthis removal, the physician views the surrounding area by fluorescentcapture to ensure that all of the malignant tissue is removed. Inparticular, the fluorophore conjugate can be used for patients who haveconditions that put the patient at increased risk for the development ofcancer. Patients who suffer this increased risk include patients withchronic inflammatory bowel disease and patients with Barrett'sesophagus.

EXAMPLE 6

This example illustrates the utility of fluorophore conjugates of thepresent invention to tumor resection procedures.

Tumor Resection:

Patients diagnosed with a cancerous lesion can be cured if the lesioncan be completely resected. Frequently, the surgeon takes tissue samplesaround the area of the resected tumor to examine the margins at the timeof surgery. If the margins appear to contain cancer cells, the surgeoncontinues to remove tissue to complete the resection. This tissueexamination helps the physician identify areas that may be precancerousor cancerous but not yet removed from the patient. Identification andremoval of these lesions prevents spread of the disease and can cure thepatient. This procedure suffers from some significant limitations, as itis time consuming, and samples from only a small area can be examined.Accordingly, many surgeons do not undertake this tissue sampling duringthe resection of the tumor but instead examine the tissue only after thesurgery is completed. If malignant tissue has been left in the patient,further therapy, such as radiation therapy, is given to the patient. Ifthe physician knew that malignant tissue remained in the patient at thetime of surgery, a more complete resection could be effected, and animproved outcome for the patient would result.

To eliminate these limitations of surgical resection of cancer,fluorophore conjugates are administered to the patient by intravenousinjection 1 to 48 hours, preferably 2 to 6 hours, prior to the tumorresection. The conjugate can alternatively be administered by oralsolution or pill, circulates in the blood and accumulates in cancerousor precancerous tissue relative to normal tissue. During the surgery,the physician views the tissue under both white light and fluorescentcapture. Specifically, the physician views an area that the physicianhas difficulty determining if it is normal or cancerous. In particular,the surgeon views the area of resected tissue prior to completing theoperation to determine that all cancerous tissue has been removed. Areasremaining that emit fluorescence and therefore are clearly malignant orpre-malignant are removed. In particular, the fluorophore conjugate isused for patients who have cancers where such definitive removal iscurrently used. This includes removal of skin cancer by the Mohs'procedure that involves examination of successively collected sectionsof tissue to determine where the cancer remains and thus where to directsubsequent resection. The use of the fluorophore conjugate can improvethe speed and ability of the surgeon to remove this type of cancer.

EXAMPLE 7

This example illustrates the utility of fluorophore glucose ordeoxyglucose conjugates of the present invention in uppergastrointestinal endoscopy.

Upper Gastrointestinal Endoscopy:

Endoscopic evaluation of the upper gastrointestinal tract is recommendedfor many patients who have chronic gastroesophageal reflux and allindividuals diagnosed with Barrett's esophagus. This screeningexamination helps the physician identify lesions that may be dysplastic,precancerous, or cancerous but still localized in the intestine.Identification and removal of these lesions prevents spread of thedisease and can cure most patients. This procedure suffers from somesignificant limitations including difficulty in distinguishing betweennormal mucosa and dysplastic or precancerous lesions that must beremoved. If the physician knew that there were areas of precanceroustissue, the patient would undergo surgical removal of the same and thelethal development of a cancer would be avoided. Due to currentlimitations, areas of precancerous tissue are often overlooked duringendoscopy. Because the physician has no aid to find the areas ofprecancerous tissue, the physician must obtain biopsies in a randomfashion, hoping to discover by chance the offending tissue if it exists.

To eliminate these limitations of endoscopy, a fluorescent conjugate ofthe invention is administered to the patient by intravenous injection 1to 48 hours, preferably 2 to 6 hours, prior to the endoscopic procedure.This compound may alternatively be given by oral solution or pill. Theconjugate circulates in the blood and accumulates in cancerous orprecancerous tissue relative to normal tissue. During endoscopy, thephysician views the esophagus under both white light and fluorescentcapture. Specifically, using fluorescent capture, the physician canidentify areas of increased fluorescence, which are deemed to beprecancerous and which can be biopsied or removed as the physician deemsappropriate.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually incorporated byreference. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for detecting or imaging cancer in a subject, said methodcomprising: (a) administering to said subject an effective amount of afluorophore glucose, deoxyglucose or derivative conjugate; and (b)detecting or imaging cells that take up said fluorophore conjugate todetermine if cancer is present in said subject.
 2. A method inaccordance with claim 1, wherein said fluorophore conjugate has theformula:Fl-L-Glc wherein Fl is a fluorophore; L is a bond or a linking group;and Glc is glucose, deoxyglucose or a glucose or deoxyglucosederivative.
 3. A method in accordance with claim 2, wherein said Glc isdeoxyglucose or a deoxyglucose derivative selected from the groupconsisting of D-(+)-deoxyglucose, D-(+)-glucosamine and N-acetylD-glucosamine, and said Fl is a fluorophore having an emissionwavelength of from about 400 nm to about 1200 nm.
 4. A method inaccordance with claim 2, wherein Fl is a fluorophore selected from thegroup consisting of nitrobenzoxazole dyes, cyanine dyes,dipyrrometheneboron dyes, coumarin dyes, benzocoumarin dyes, xanthenedyes, benzo[a]xanthene dyes, benzo[b]xanthene dyes, benzo[c]xanthenedyes, phenoxazine dyes, benzo[a]phenoxazine dyes, benzo[b]phenoxazinedyes and benzo[c]phenoxazine dyes.
 5. A method in accordance with claim2, wherein L is a linking group having from 2 to 30 chain atoms selectedfrom the group consisting of C, N, O, P, S and Si.
 6. A method inaccordance with claim 2, wherein said fluorophore glucose ordeoxyglucose conjugate has a formula selected from the group consistingof:

wherein Fl is a fluorophore; L is a linking group; each X isindependently selected from the group consisting of O and NH; R¹, R³ andR⁴ are each members independently selected from the group consisting ofH, (C₁-C₁₂)alkyl, (C₁-C₁₂)acyl and a solubility or partitioning effectorcomponent.
 7. A method in accordance with claim 1, wherein saiddetecting or imaging is carried out in combination with a cancerresection.
 8. A method in accordance with claim 1, wherein said cancercells are selected from the group consisting of lung cancer cells,breast cancer cells, prostate cancer cells, colon cancer cells, cervicalcancer cells, esophageal cancer cells, bladder cancer cells, head andneck cancer cells and melanoma cells.
 9. A method in accordance withclaim 1, further comprising a preliminary step of reducing glucoseingestion in said subject.
 10. A method of detecting pre-cancerous cellsin a subject, said method comprising: (a) administering to said subjectan effective amount of a fluorophore glucose or deoxyglucose conjugate;and (b) detecting or imaging pre-cancer cells that take up saidfluorophore conjugate to determine if pre-cancer cells are present insaid subject.
 11. A method in accordance with claim 10, wherein saidfluorophore conjugate has the formula:Fl-L-Glc wherein Fl is a fluorophore; L is a bond or a linking group;and Glc is glucose, deoxyglucose or a glucose or deoxyglucosederivative.
 12. A method in accordance with claim 11, wherein Fl is afluorophore selected from the group consisting of nitrobenzoxazole dyes,cyanine dyes, dipyrrometheneboron dyes, coumarin dyes, benzocoumarindyes, xanthene dyes, benzo[a]xanthene dyes, benzo[b]xanthene dyes,benzo[c]xanthene dyes, phenoxazine dyes, benzo[a]phenoxazine dyes,benzo[b]phenoxazine dyes and benzo[c]phenoxazine dyes
 13. A method inaccordance with claim 10, wherein said administering is intravenous,oral or topical.
 14. A method in accordance with claim 13, wherein saidpre-cancerous cells are associated with Barrett Esophagus, colonicpolyps and inflammatory bowel disease.
 15. A fluorophore conjugatehaving the formula:Fl-L-Glc wherein Fl is a fluorophore having an emission wavelength offrom about 400 nm to about 1200 nm; L is a bond or a linking group; andGlc is glucose, deoxyglucose or a glucose or deoxyglucose derivative,with the proviso that said fluorophore deoxyglucose conjugate is otherthan 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose. 16.A fluorophore conjugate of claim 15, having a formula selected from thegroup consisting of:

wherein Fl is a fluorophore; L is a linking group; each X isindependently selected from the group consisting of O and NH; R¹, R³ andR⁴ are each members independently selected from the group consisting ofH, (C₁-C₁₂)alkyl, (C₁-C₁₂)acyl and a solubility or partitioning effectorcomponent.
 17. A fluorophore conjugate of claim 16, wherein Fl isselected from the group consisting of coumarins, xanthenes, phenoxazinesand cyanines.
 18. A method for cancer or pre-cancer detection during anoperative or endoscopic procedure, said method comprising: (a)administering to a patient subject to said procedure, an effectiveamount of a fluorescent glucose or deoxyglucose conjugate, saidconjugate having a rate of uptake in cancerous or pre-cancerous cellsthat is at least two times greater than the rate of uptake in normalcells; (b) conducting said procedure within 48 hours of saidadministering; (c) scanning said patient with a detection means fordetecting the localization of said fluorescent conjugate in said canceror pre-cancer cells.
 19. A method in accordance with claim 18, whereinsaid procedure is conducted within 24 hours of said administering and iseither an operative procedure or an endoscopic procedure, and whereinsaid method further comprises treating sites of conjugate accretion byexternal beam radiation, laser therapy, or surgical removal.
 20. A kitcomprising a vial containing a sterile preparation for human use of afluorophore glucose, deoxyglucose or derivative conjugate and apharmaceutically acceptable carrier.